Viral inactivation or removal is required for the production process of an antibody drug containing a monoclonal antibody produced by cell culture, because of concerns about contamination with viruses from raw materials or production steps. As a method for inactivation of viruses that may contaminate an antibody drug, heat treatment, treatment using a chemical agent or the like is performed. However, viruses cannot be sufficiently inactivated by such treatment alone. Also, these methods may directly denature the antibody in the antibody drug. Starting from this background, viral separation and removal using filter membranes are performed as a physical means for removing viruses without chemical denaturation.
As filter membranes for viral removal, a membrane comprising natural material such as cellulose and a virus-removing membrane comprising a synthetic polymer material such as polyvinylidene fluoride (PVDF) or polyether sulfone (PES) are known (Non-Patent Documents 1-4).
Ideally, a larger amount of an antibody can be filtered within a short time and viruses can be removed with sufficiently high virus removal performance through the filtration of an antibody drug using a virus removal device that includes the above virus-removing membrane. However, in actuality, a cellulose membrane is problematic in that it tends to become clogged even at an antibody concentrations of 20 mg/ml or higher, exhibits low pressure resistance, and can increase actual working pressure to only about 100 kPa, although filtration is possible, for example. Alternatively, a synthetic polymer membrane may have high pressure resistance and may function without problems even if the actual working pressure is increased to about 300 kPa. However, the synthetic polymer membrane is problematic in that it becomes clogged when the antibody concentration is increased to about 20 mg/ml, making filtration impossible to perform. Hence, filtration is generally performed at low concentrations of 10 mg/ml or lower.
However, in recent years, the pharmaceutical concentrations of antibody drugs have been on the increase. Reflecting the trend, the demand for an increase in antibody concentration during the filtration step for removing viruses is increasing. When the antibody concentration in a monoclonal antibody solution is increased, monoclonal antibodies tend to become associated with each other so as to form aggregates. When filtration is performed using a membrane having a small pore diameter, as in the case of a virus-removing membrane, association of monoclonal antibodies with each other becomes further significant because of physical stresses resulting from filtration, and thus the virus-removing membrane becomes clogged as described above.
In particular, in order to remove a small virus having a diameter of about 18-24 nm such as a parvovirus from a monoclonal antibody solution at a high removal rate, a virus-removing membrane with a small pore diameter intended for the removal of parvoviruses is required. Such a membrane is problematic in that it becomes easily clogged when a high concentration monoclonal antibody solution is filtered, the resulting antibody recovery rate is disadvantageously low, and a very long time is required for filtration.
There is a prior art reference that does not disclose any monoclonal antibody, but discloses a method for removing viruses from a protein solution by nanofiltration. Specifically, the method targeting fibrinogen comprises: adding at least one ingredient that is selected from a chaotropic substance selected from arginine, guanidine, citrulline, urea, a derivative thereof, and a salt thereof, and a compound selected from polyethoxy sorbitan ester and a derivative thereof, to a protein solution; and then filtering the protein solution using a virus-removing membrane having a pore diameter that is 15 nm or more and less than 35 nm (Patent Document 1).
Patent document 1 discloses the assumption that the ingredient may suppress or inhibit the association of protein molecules or hydrated layer formation in the vicinity of molecules. However, intended proteins herein are blood coagulation factors such as fibrinogen and VIII factor. Also, in examples in Patent Document 1, the membrane permeability of a fibrinogen solution in the presence of arginine is merely compared with the same in the absence of arginine. Furthermore, the fibrinogen concentration is less than 5 mg/ml and the subject is a low-concentration solution. Fibrinogen is a long, slender, thread-shaped protein having a length of nearly 60 nm, which is polymerized upon bleeding and thus is useful for hemostasis. On the other hand, a monoclonal antibody is a spherical protein having a diameter of about 15 nm and having physicochemical properties (e.g., isoelectric point and hydrophilicity) that differ significantly from those of fibrinogen. Patent Document 1 is an invention relating to fibrinogen. Moreover, the invention of Patent Document 1 is not a technology relating to monoclonal antibodies, but a technology relating to fibrinogen as a protein having properties completely differing from those of monoclonal antibodies. Thus, Patent Document 1 is not a good reference for the purification of monoclonal antibodies.
Patent Document 2 describes a method for removing viruses from a fibrinogen-containing solution that may contain viruses by using a virus-removing membrane, which is characterized in that the solution containing fibrinogen contains basic amino acid or salts thereof and sodium chloride. Patent Document 2 also relates to viral removal using a membrane wherein a fibrinogen solution is used. Moreover, the protein concentration in Patent Document 2 ranges from as low as 5 mg/ml to 16.5 mg/ml, significantly differing from the high concentration monoclonal antibody solution that is an object of the present application. Furthermore, the virus-removing membrane used in Patent Document 2 is a membrane with low ability to remove small viruses such as parvoviruses, and it allows small viruses to pass through it. The sizes of viruses to be removed by the invention of Patent Document 2 are larger than those of the subject viruses of the present application. Hence, the technology of Patent Document 2 poses no problem upon filtration concerning the relationship between aggregates of monoclonal antibodies and the membrane.
Solution conditions (e.g., pH and ionic strength) when a virus-removing membrane is used in a purification process for a monoclonal antibody, are varied. Accordingly, the physicochemical properties of the antibody surface and the membrane surface differ depending on solution conditions. Actually, there has been a case in which the flux was very low upon antibody filtration depending on solution conditions. The interaction between the antibody surface and the membrane surface is one reason for such a low flux, and in particular, electrostatic interaction that functions between the two affects such a low flux. The electric charge property of the antibody surface and the membrane surface is expressed as surface potential (zeta potential) that is changed to a positive or negative potential state depending on the relationship between the solution pH and isoelectric point (pI). It is known that pI of a monoclonal antibody ranges from 6 to 10. When pH<pI, a monoclonal antibody has high positive potential and acts adversely in membrane filtration. Therefore, it is thought that if the surface potential of an antibody is lowered and its electrostatic interaction with the membrane is suppressed, the flux during filtration will be improved. Meanwhile, under such solution conditions, a high concentration monoclonal antibody solution is problematic in that dispersion stability becomes poor because of the antibodies' own charges, antibodies tend to form aggregates, and thus the flux decreases over time during membrane filtration.
Specifically, there has been no prior art concerning a method for removing even small viruses using a membrane within a short time at high yield from a high concentration monoclonal antibody solution through the control of the surface potentials of the membrane and antibodies and the suppression of the association of antibodies (contained in a solution at a high concentration) with each other, so as to improve the filterability of the membrane.